Study of AP endonuclease (APEX1/REF1), a DNA repair enzyme, in gallbladder carcinoma.

AIM: This study investigated the levels of Apurinic/Apyrimidinic Endonuclease (APEX1) in gallbladder carcinoma (CaGB) tissue and co-related these levels with various clinicopathological parameters. PATIENTS AND METHODS: Twenty cases of CaGB and cholelithiasis were included in the study. Western blot analysis of APEX1 protein was performed using actin as the reference point. Densitometric analysis and the integrated density value (IDV) of APEX1 protein samples were determined. The ratio of IDV of APEX1/actin was determined. RESULTS: The mean IDV ratio of APEX1 in CaGB was 0.63±0.33 and 0.45±0.19 in cholelithiasis. The mean IDV ratio of a variant of APEX1 (ΔAPEX1) in CaGB was 0.50±0.09, whereas it was 0.40±0.16 in cholelithiasis. Calculating the mean IDV ratio of total APEX (APEX1+ΔAPEX1) in CaGB was 1.13±0.31 whereas in cholelithiasis, 0.85±0.23. The differences were statistically significant (p<0.05). CONCLUSION: A significant correlation was found between the relative expressions of APEX1 in cancer as compared to that in cholelithiasis patients. There was significant association between APEX1 expression and perineural invasion. A variant of APEX1 correlated with tumor infiltration. Hence APEX1 may be of use as a prognostic marker in patients with CaGB.

Effect of 5-fluorouracil and mitomycin on the healing of intestinal anastomosis.

OBJECTIVE: Antineoplastic agents affect the healing of intestinal anastomosis. The aim was to evaluate the effect of 5-fluorouracil (5-FU) and mitomycin on the healing of the intestinal anastomosis and their schedule of administration. MATERIAL AND METHODS: Eighty-nine male albino Charles Foster rats with a mean weight of 256.57 g were divided into six groups. Group A represents the control, while in others varying schedules of chemotherapy (5-FU and mitomycin) were administered. The sacrifices were made on days 7, 14 and 21 postoperatively and bursting pressure and hydroxyproline content were measured. RESULTS: Nine rats died before completion of the experiment and were excluded. Adhesions were noted in all rats on sacrifice. The mean bursting pressure of normal intestine (group A) was 252 mm Hg. The bursting pressure was lower on day 7 (208 mm Hg) and it subsequently increased by day 21 (230 mm Hg). The mean bursting pressure in groups B, C, D and E was 174, 194, 182 and 188 mm Hg and it subsequently increased to 232, 272, 244 and 286 mm Hg. There was no difference in the pattern of bursting pressure in colon and ileum. The mean hydroxyproline content of ileum (group A) on day 7 was 34.37 mg/g tissue. The hydroxyproline content of the ileum in groups B, C, D and E was 15.08, 27.03, 7.75 and 21.04 mg/g tissue. There was a significant decrease in hydroxyproline content following anastomosis and chemotherapy. CONCLUSIONS: The effect of chemotherapy was pronounced when administered on the day of surgery or in the immediate pre- or postoperative period. Hence administration of chemotherapy during this period may be harmful.

Alteration of catecholamines in pheochromocytoma (PC12) cells in vitro by the metabolites of chlorotriazine herbicide.

The effects of four major chlorotriazine metabolites on the constitutive synthesis of the catecholamines dopamine (DA) and norepinephrine (NE) were examined, using undifferentiated PC12 cells. NE release and intracellular DA and NE concentrations were quantified, for up to 24 h after initiation of treatment with different concentrations, ranging from 0 to 400 microM, of the metabolites hydroxyatrazine (HA), 2-amino-4-chloro-6-isopropylamino-s-triazine (deethylchlorotriazine), 2-amino-4-chloro-6-ethylamino-s-triazine (deisopropylchlorotriazine), and diaminochlorotriazine (DACT). Hydroxyatrazine significantly decreased intracellular DA and NE concentrations in a dose- and time-dependent manner. This metabolite also caused a significant inhibition of NE release from the cells. In contrast, deethylchlorotriazine and deisopropylchlorotriazine significantly increased intracellular DA concentration following exposure to 50-200 microM from 12 to 24 h. Intracellular NE was significantly reduced at these same concentrations of deethylchlorotriazine at 24 h while the concentration of NE in PC12 cells exposed to deisopropylchlorotriazine was not altered at any dosage or time point measured. NE release was decreased at 18 (200 microM) and 24 h (100 and 200 microM) following exposure to deethylchlorotriazine and at 24 h (100 and 200 microM) following deisopropylchlorotriazine. DACT, at the highest concentration (160 microM), significantly increased intracellular DA and NE concentrations at 18 and 24 h. NE release was also increased at 40-160 microM at 24 h. The viability of the PC12 cells was tested using the trypan blue exclusion method. Following 18 to 24 h of treatments with HA, cell viability was reduced 10-12% at the two higher concentrations (200 and 400 microM), but, with other metabolites, the viability was reduced by only 2 to 5% at the highest concentrations. These data indicate that HA affects catecholamine synthesis and release in PC12 cells in a manner that is similar to synthesis of atrazine and simazine. On the other hand, deethylchlorotriazine and deisopropylchlorotriazine altered catecholamine synthesis in a manner similar to that observed in the rat brain following in vivo exposure (i.e., increased DA and decreased NE concentration), whereas DACT appeared to produce an increase in NE release as well as in the intracellular DA and NE concentrations. Overall, these findings suggest that the catecholamine neurons may be a target for the chlorotriazines and/or their metabolites, that the metabolites produce a unique pattern of catecholamine response, and that all of the changes were seen within the same range of doses.

Differential modulation of catecholamines by chlorotriazine herbicides in pheochromocytoma (PC12) cells in vitro.

Epidemiological, wildlife, and laboratory studies have pointed to the possible adverse health effects of chlorotriazine herbicide (i.e. , atrazine, simazine, and cyanazine) exposure. However, the cellular mechanism(s) of action of these compounds remains unknown. Recently, it was reported by Cooper et al. (2000, Toxicol. Sci. 53, 297-307) that atrazine disrupts ovarian function by altering hypothalamic catecholamine concentrations and subsequently the regulation of luteinizing hormone (LH) and prolactin (PRL) secretion by the pituitary. In this study, we examined the effect of three chlorotriazines on catecholamine metabolism in vitro using PC12 cells. Intracellular norepinephrine (NE) and dopamine (DA) concentrations and spontaneous NE release were measured following treatment with different concentrations of atrazine, simazine (0, 12. 5, 25, 50, 100, and 200 microM) and cyanazine (0, 25, 50, 100, and 400 microM) for 6, 12, 18, 24, and 48 h. Atrazine and simazine significantly decreased intracellular DA concentration in a concentration-dependent manner. Intracellular NE concentration was also significantly decreased by 100 and 200 microM atrazine and 200 microM simazine. Similarly, there was a dose-dependent inhibition of NE release with 100 and 200 microM concentrations of both compounds. Although 100 and 400 microM cyanazine increased intracellular NE concentration, 50, 100, and 400 microM cyanazine significantly increased NE release at 24 and 36 h. In contrast, intracellular DA concentration was decreased by cyanazine, but only at 400 microM. The GABA(A)-receptor agonist, muscimol (0, 0.01, 0.1, and 1.0 microM) had no effect on either the release or on intracellular catecholamine concentrations from 6 through 24 h of treatment. Cell viability was somewhat lower in the groups exposed to 100 and 200 microM atrazine and simazine. However, the reduction in viability was significant only in the highest dose of atrazine used (200 microM) at 24 h. Cyanazine did not have an effect on the viability at any of the doses tested, and the cells were functional, even up to 48 h of exposure. These data indicate that both atrazine and simazine inhibit the cellular synthesis of DA mediated by the tyrosine hydroxylase (TH), and NE mediated by dopamine beta-hydroxylase (DbetaH), and, as a result, there is a partial or significant inhibition of NE release. Cyanazine, on the other hand, stimulated the synthesis of intracellular NE, and not DA. Thus, chlorotriazine compounds presumably act at the enzymatic steps or sites of CA biosynthesis to modulate monoaminergic activity in PC12 cells.

Dried sera for confirming blood-borne virus infections (HCV, HTLV-I, HIV & HBsAg).

For safe blood transfusion, developing countries face considerable problems including serological screening and confirmation of blood-borne virus infections (HCV, HTLV-I, HIV and HBsAg). Confirmation tests are not only costly but also require sophisticated techniques and expertise. In order to provide this support we have attempted to perform a virus antibody confirmation test on samples dried on blotting paper (BP). Forty-nine sera derived from selected patients and donors from Bombay, and nine donors' sera from Bellarussia were transported on BP. In control experiments, dilutions of antibody-positive sera (HIV, HTLV-I & HCV) and 'blinded' HTLV-I antibody-positive and antibody-negative donors were applied on BP. Eluates from snipped BP were tested initially by screening tests, and the reactives were subjected to confirmatory tests for three types of virus antibody tests (HCV, HTLV-I & HIV) by blotting methods and neutralisation tests for HBsAg. There was considerable reduction of titres in dry sera but all BP-derived dry specimens gave excellent qualitative concordance with their liquid-equivalent sera, and the HTLV-I-positive donor was identified and reconfirmed correctly. Presence of only HCV antibody was confirmed in all the nine selected Bellarussian donors. Blood donors in Bombay had 3% HIV antibody, 6% HBsAg and none had HCV antibody, while selected patients showed substantially higher levels of these markers: HIV-antibody 64%, HBsAg 57% and HCV-antibody 17% confirmed positive. The cause of this high level remains to be established. Dry samples received by post seem to be an economical approach to a first step in providing some levels of independent confirmation of reactives in developing countries.

Rectal stricture: a complication of tuberculosis.

Tuberculosis of the rectum is rarely reported, even from areas where tuberculosis, and gastrointestinal tuberculosis in particular, is prevalent. The authors report a case of long tubercular stricture of the rectum and distal part of the sigmoid colon in a 12-year-old girl. Because of nonspecific symptoms and noncharacteristic radiological and endoscopic features, the diagnosis of this rare entity rests mainly on histological evidence of the classical tubercle in a surgical biopsy specimen.

Cystic teratoma of the pancreas.

Cystic teratoma of the pancreas is an extremely rare entity: only 11 cases have been described so far in the world literature. We report the 12th case, in a 4-month-old girl.

Activation of resident tissue-specific macrophages by swainsonine.

The induction of macrophage tumoricidal activity by swainsonine (8a beta-indolizidine-1 alpha, 2 alpha, 8 beta-triol), an indolizidine alkaloid, has been implicated as possibly an important immune effector mechanism involved in the suppression of tumor growth and metastasis in vital organs such as the lung, liver and spleen (Olden, K. et al. The potential importance of swainsonine in therapy for cancers and immunology. Pharmacol. Ther. 50:285-290; 1991). The present study further explores this possibility by determining whether resident tissue-specific macrophages of several mouse strains can be rendered tumoricidal by systemic administration of swainsonine. We found that systemically administered swainsonine could increase the tumoricidal activity of both alveolar (lung) and splenic macrophages. The activity was enhanced as much as 3- to 4-fold over that obtained with macrophages from organs of control animals and was both dose- and time-dependent. The level and extent of activation by swainsonine was comparable to that achieved with traditional macrophage-activating agents, such as lipopolysaccharide and interferon-gamma. The fact that swainsonine activated highly purified (> 95%) cultures of macrophages from the various sources suggests a direct mechanism of activation. Furthermore, the in vivo activation of macrophages in immune-compromised animals (SCID and nude) lends credence to this suggestion. These findings provide a plausible explanation for the observations that systemically administered swainsonine inhibits organ colonization of metastatic cells and growth of SC tumor xenografts, whereas the growth of tumor cells is not inhibited by swainsonine in culture.

Retrograde intussusception: a lead-point for prograde intussusception.

Retrograde intussusception is an extremely rare entity. The authors report a case of retrograde jejuno-jejunal intussusception in a 12 year old. The features of interest are (1) the retrograde intussusception mass acting as a lead-point for a second prograde jejuno-jejunal intussusception, (2) the anastomotic suture line of a previous resection-anastomosis acting as the lead-point for the retrograde intussusception, and (3) the presence of a "reverse claw sign" (on a barium meal study) caused by the intussusceptum of the retrograde intussusception.

Recovery of fibrinogen in cryoprecipitate pasteurized in the presence of sucrose and glycine

The effect of sucrose (60 per cent w/w) and 1 M glycine as thermal stabilizers for fibrinogen in cryoprecipitate was studied. Sucrose (9.2 g) and glycine (0.9 g) were dissolved in 6 g of cryoprecipitate and the solution was pasteurized at 60 degrees C for 10 h. The preparation was then dialyzed for 20 h in phosphate buffered saline (PBS), lyophilized, stored for one week at -40 degrees C and resuspended in distilled water. The recovery of total proteins and fibrinogen in the final product averaged 66.4 +/- 4.1 per cent and 43.8 +/- 6.4 per cent of the initial contents, respectively (mean +/- SEM, N = 9). The pasteurization of cryoprecipitate in the presence of PBS (control experiments) produced extensive precipitation, which is characteristic of protein denaturation. Thus, this method partially protected fibrinogen and other proteins in cryoprecipitate from inactivation by prolonged exposure to heat during pasteurization

Spontaneous umbilical fistula: a rare complication of ventriculoperitoneal shunt.

Although complications following ventriculoperitoneal shunt for hydrocephalus are not uncommon, leak of cerebrospinal fluid from the normal umbilicus following ventriculoperitoneal shunt is very rare. One such case is reported.

Serotonergic-cholinergic neurotransmitters' function in brain during cadmium exposure in protein restricted rat.

Daily subcutaneous injection of cadmium chloride (0.3 mg/100g bw) for two weeks to normal and protein restricted (5% casein) rats shows significant decrease in 5-HT concentration in cerebellum, medulla oblongata-pons, hypothalamus, striatum-hippocampus, midbrain-thalamus-subthalamus, and cortex in both dietary regimens. No significant change occurs in concentration of ACh in cerebellum, but there is a significant increase in cortex, whereas significant decrease occurs in rest of the discrete regions of brain in both dietary conditions. Results also indicate that the intensity of cadmium effect is more evident in discrete brain regions in protein restricted dietary condition than in the normal group. The inhibitory action of Cd on both neurotransmitters has been discussed.

A simplified method for purification, concentration and freezing of bone marrow cells for autologous transplantation.

Autologous bone marrow (BM) transplantation is being increasingly applied in hematological and oncological patients. However, because of the need to purify and preserve BM requiring high technology, such treatments are virtually concentrated in the "developed" countries. This paper examines methods of BM purification and freezing that could make the technique potentially applicable in developing countries. Hemapheresis is routinely applied for BM purification in our Dutch Centre, where the buffy coats obtained from routine blood donations were utilised in experimental settings. Using DMSO as cryoprotectant, semi-purified white cells were frozen in liquid N2 (LN2), by mechanical freezer or snap-frozen at -55 degrees C. Different types of containers were compared including plastic tubes and ordinary blood bags. After thawing the results show that snap-freezing had a deleterious effect but the cell yields and viability were similar in LN2 or the mechanical freezer where the tubes and the bag were equally effective as containers (86% cell recovery with 90% viability). In the purification/concentration stage, reduction of the volume of the material by extra centrifugation, thus requiring less DMSO, produced better results--96% cell yield and 90% viability after thawing. This simplified method was applied in a general hospital in Sao Paulo where four oncology patients underwent BM collection. BM was purified and concentrated within a blood bank facility. Hydroxyethyl starch sedimentation and centrifugation of the material in plastic blood bags gave 80% BM cell harvest. After thawing from the mechanical freezer 1 x 10(8) BM cells/kg were available for reinfusion to patients. There was no immediate untoward reaction. Three of the patients showed signs of bone marrow regeneration by three weeks, but one patient died 16 days after transplantation, of septicemia. We conclude that certain high-technology procedures for ABMT can be adapted for existing facilities in developing countries.